The use of confocal laser scanning microscopy to analyze the process of parasitic protozoon-host cell interaction.

In this communication we review the results obtained with the confocal laser scanning microscope to characterize the interaction of epimastigote and trypomastigote forms of Trypanosoma cruzi and tachyzoites of Toxoplasma gondii with host cells. Early events of the interaction process were studied by the simultaneous localization of sites of protein phosphorylation, revealed by immunocytochemistry, and sites of actin assembly, revealed by the use of labeled phaloidin. The results obtained show that proteins localized in the interaction sites are phosphorylated. The process of formation of the parasitophorous vacuole was monitored by labeling the host cell surface with fluorescent probes for lipids (PKH26), proteins (DTAF) and sialic acid (FITC-thiosemicarbazide) before interaction with the parasites. Evidence was obtained indicating transfer of components of the host cell surface to the parasite surface in the beginning of the interaction process. We also analyzed the distribution of cytoskeletal structures (microtubules and microfilaments visualized with specific antibodies), mitochondria (visualized with rhodamine 123), the Golgi complex (visualized with C6-NBD-ceramide) and the endoplasmic reticulum (visualized with anti-reticulin antibodies and DIOC6) during the evolution of intracellular parasitism. The results obtained show that some, but not all, structures change their position during evolution of the intracellular parasitism.

Effect of protein kinase inhibitors on the invasion process of macrophages by Trypanosoma cruzi.

The interaction of Trypanosoma cruzi with different vertebrate cells involves two distinct steps, attachment and internalization. Genistein and staurosporine, drugs which inhibit protein kinases, specially tyrosine kinase, are able to block the infection of macrophages by T. cruzi, suggesting that protein phosphorylation is a key event on this process.

Interaction of Trypanosoma cruzi with cells with altered glycosylation patterns.

Glycosylation mutants of chinese hamster ovary cells were used to analyse the role played by surface-exposed carbohydrates on the process of interaction of Trypanosoma cruzi with the host cell. Adhesion and invasion of the parasites were markedly reduced in cells which express very few sialic acid residues. Infection levels similar to those obtained with the parental cell could be obtained after sialylation of the mutant cell using exogenous fetuin as sialic acid donor and T. cruzi trans-sialidase. The results obtained show that host cell sialic acid residues are involved in the process of attachment to and penetration of T. cruzi into the host cell.

Penetration of Toxoplasma gondii into host cells induces changes in the distribution of the mitochondria and the endoplasmic reticulum.

Fluorescence microscopy, using dyes which specifically label mitochondria, endoplasmic reticulum and the Golgi complex, and transmission electron microscopy, were used to analyze the changes which occur in the organization of these structures during interaction of Toxoplasma gondii with host cells. In uninfected cells the mitochondria are long filamentous structures which radiate from the nuclear region toward the cell periphery. After parasite penetration they become shorter and tend to concentrate around the parasite-containing vacuole (parasitophorous vacuole) located in the cytoplasm of the host cell. The mitochondria of extracellular parasites, but not of those located within the parasitophorous vacuole, were also stained by rhodamine 123. Labeling with DiOC6, which binds to elements of the endoplasmic reticulum, in association with transmission electron microscopy, revealed a concentration of this structure around the parasitophorous vacuole. The membrane lining this vacuole was also stained, suggesting that components of the endoplasmic reticulum are also incorporated into this membrane. The Golgi complex, as revealed by staining with NBD-ceramide and electron microscopy, maintains its perinuclear position throughout the evolution of the intracellular parasitism.

Inhibitory effect of melanin on the interaction of Fonsecaea pedrosoi with mammalian cells in vitro.

Conidial forms of Fonsecaea pedrosoi, grown under conditions where melanin was or was not synthesized, were allowed to interact with normal and cytochalasin treated macrophages. Melanin-free conidia were more infective to the macrophages. Treatment of macrophages with either cytochalasin B or D before the interaction decreased, but did not totally prevent their infection by the fungi. This inhibitory effect was higher (approximately 90%) if F. pedrosoi was grown under conditions where melanin was not synthesized. When melanin-containing conidia were used, the inhibitory effect of the cytochalasin on the infection was lower (approximately 50%). At least two mechanisms of infection of the host cell were observed: typical phagocytosis and another process in which the fungi played a more active role. Infection by F.pedrosoi was also observed in the non-professional phagocytic MDCK epithelial cell line. Two types of cytoplasmic vacuoles which contained parasites were seen in thin sections of host cells infected with F.pedrosoi: a 'tight' type and a 'loose' type. At least 200 conidia-containing vacuoles were analysed by transmission electron microscopy. The 'tight' type was observed in 75% of the vacuoles of non-treated macrophages, suggesting an association with classical phagocytosis. On the other hand, the 'loose' type vacuole was seen in 75% of the vacuoles present in cytochalasin treated macrophages and seemed to be related to induced phagocytosis or active penetration by the fungi.

Fine structure and cytochemistry of the interaction between Fonsecaea pedrosoi and mouse resident macrophages.

The interaction between conidia of Fonsecaea pedrosoi and mouse resident peritoneal macrophages was observed by light microscopy and by scanning (SEM) and transmission (TEM) electron microscopy. The conidia first attached to the surface of the macrophage and were then ingested. Prolonged incubation of the macrophage cultures showed proliferation of intracellular fungi as well as those which remained attached to the macrophage surface. The conidia were ingested by a typical phagocytic process, with formation of a phagosome. Macrophage lysosomes were observed to fuse with the phagosomes by immunofluorescence microscopy of macrophages previously labeled with acridine orange, by TEM of thin sections of macrophages labeled with albumin-gold, and by ultrastructural localization of acid phosphatase within the phagosomes.

Study of mitochondrial organization in living resident and activated macrophages using the laser dye rhodamine 123.

Rhodamine 123, a fluorescent laser dye that labels metabolically active mitochondria of living cells, was used to analyse the pattern of distribution of mitochondria in resident and activated mouse peritoneal macrophages kept in culture for 4 or 24 hr. Activated macrophages kept for 4 hr in culture showed abundant small mitochondria distributed throughout the cell. This pattern changes to a situation in which the mitochondria are filamentous and radiate from the perinuclear region when these cells are kept in culture for 24 hr, acquiring a pattern similar to that observed in resident macrophages. After treatment with phorbol myristate acetate, resident macrophages presented a mitochondrial distribution similar to that observed in activated macrophages.

The use of albumin-gold to follow lysosome-phagosome fusion.

A new procedure for labeling of secondary lysosomes and to determine, by transmission electron microscopy, their fusion with phagosomes was developed. It is based on the use of albumin adsorbed to colloidal gold particles as a probe and tested using macrophages previously labeled with albumin-gold and then incubated in the presence of epimastigote forms of Trypanosoma cruzi.

Effect of phorbol-12-myristate-13-acetate (PMA) on the fine structure of Trypanosoma cruzi and its interaction with activated and resident macrophages.

Epimastigotes of Trypanosoma cruzi showed many filopodium-like projections from the flagellar membrane when incubated in the presence of 100 ng/ml (a concentration which does not interfere with cell motility and viability) phorbol-12-myristate-13-acetate (PMA). PMA is a substance which binds to the membrane-associated protein kinase C. Few of these projections were observed in control parasites. PMA, when added to the interaction medium, significantly increased the attachment of epimastigotes to the surface and their ingestion by resident or activated mouse peritoneal macrophages maintained in vitro. PMA, however, did not interfere with the ingestion of trypomastigotes.

Cytochemical localization of NADH and NADPH oxidases during interaction of Trypanosoma cruzi with activated macrophages.

NADH- or NADPH-oxidase activity was cytochemically detected at the ultrastructural level during the process of interaction between Trypanosoma cruzi and activated mouse peritoneal macrophages. The reaction product, indicative of enzyme activity, was found in the portion of the plasma membrane of the macrophages to which the parasites attached. It was also found in the membrane which surrounds endocytic vacuoles containing ingested parasites.

Trypanosoma cruzi: surface charge and freeze-fracture of amastigotes.

Amastigotes of Trypanosoma cruzi, within vertebrate cells or isolated from the supernatant of vertebrate cell cultures (L-A9 fibroblast or J774G8 macrophage-like cell lines), possess glycoproteins or glycolipids on the cell surface according to the periodic acid-thiosemicarbazide-silver proteinate technique used in association with electron microscopy. The cell surface of isolated amastigotes is negatively charged, as evaluated by the binding of cationic particles (colloidal iron hydroxyde at pH 1.8 and cationized ferritin at pH 7.2) as well as by direct measurement of cellular electrophoretic mobility. Amastigotes (Y strain) isolated from the spleen of infected mice and amastigotes (Y and CL strains) from the supernatant of cell cultures previously infected with T. cruzi have the same mean electrophoretic mobility (-0.85 micron sec-1 V-1 cm). It is intermediate between the epimastigote and the trypomastigote forms (determined previously). Sialic acid is the important component responsible for the negative surface charge, as determined by the use of neuraminidase. Thus, it is possible to use the mean electrophoretic mobility as an indicator for identifying amastigotes of T. cruzi.

Further studies on the cell surface charge of Trypanosoma cruzi.

Trypanosoma cruzi has a negative surface which varies according to the ionic strength and the pH of the solution in which the cells are suspended. At low pH there is a decrease in the negative surface charge with an isoelectric point at pH 2.6 and 3.0 for epimastigote and trypomastigote forms, respectively. Below these pH values the cells have a positive surface charge. At higher pH there is an increase in the surface charge. Glutaraldehyde fixation did not interfere with the electrophoretic mobility (EPM) of the cells. Epimastigote and trypomastigote forms of T. cruzi have a characteristic EPM independent of the medium in which the cells were grown, the origin of the trypomastigotes or the strain of the parasite. Trypomastigotes have a higher negative surface charge than epimastigotes. Based on the change in the EPM of the cells treated with neuraminidase, is concluded that sialic acid is present on the cell surface of T. cruzi and that it is the main component responsible for the high negative surface charge of the trypomastigote form. Trypsin treatment also reduces the EPM of T. cruzi. Neuraminidase or trypsin-treated parasites recovered their normal EPM when incubated for 4 h in fresh culture medium. This process involves synthesis of protein since it is inhibited by puromycin.

Separation of amastigotes and trypomastigotes of Trypanosoma cruzi from cultured cells.

A method is described for the isolation and purification of trypomastigotes and amastigotes of Trypanosoma cruzi from cell cultures. L-A9, a transformed fibroblast cell line, and J774G8, a macrophage-like cell line of tumor origin, were used. Both cell lines were infected with bloodstream trypomastigotes of T. cruzi, which once within host cells transform into dividing amastigotes. After 6--8 days infection the host cells ruptured, spontaneously liberating parasites into the culture medium. L-A9 cells liberated mainly trypomastigotes while J774G8 cells liberated amastigotes. The parasites were collected and purified by centrifugation in a gradient of metrizamide. The purity of the preparation as well as the morphology of the parasites and the host cells were analysed by electron microscopy.